1. Field of the Invention
The present invention relates to a phosphorylating agent and a method for forming a phosphodiester bond using the phosphorylating agent.
2. Prior Art
The phosphodiester bond is universally present in many biomolecules such as nucleic acids, phospholipids, sugar phosphates and coensymes. These molecules are essential for organisms to manifest various functions thereof and to preserve the species by themselves. Recently, there have been actively done many attempts to imitate the excellent functions of these biomolecules and to make the most use of knowledges about organic chemistry to thereby apply them in chemical and technological fields. For this reason, it has become one of most important subjects in recent organic synthesis to synthesize molecules having a phosphodiester bond in high efficiency.
The simplest and most rational method for forming a phosphodiester bond generally comprises phosphorylating two kinds of alcohol components in order with a divalent phosphorylating agent. Phosphorylating agents often used for this purpose are chlorophosphates such as phenylphosphoric acid dichloride (see, for instance, Chemistry and Physics of Lipids, 41, 1980, p. 1). In particular, phenylphosphoric acid dichloride is presently put on the market and the method for phosphorylating two kinds of alcohols in order with the reagent has been used as a simple and reliable technique since the earliest stage of studies in this field, for example, in the synthesis of nucleic acids and phospholipids. However, this phosphorylating agent is a bifunctional reagent carrying two chlorine atoms as leaving groups and it does not show any difference in reactivity between the first and second phosphorylation reactions. For this reason, it suffers from a severe problem in that these reactions are inevitably accompanied by formation of undesirable symmetrical phosphates as by-products. For instance, this method was used in the studies of nucleic acid synthesis in the early stage, and, in 1978, Reese et al. reported the synthesis of TpT using phenylphosphoric acid dichloride, but it has been pointed out that the method likewise suffers from the problem of the formation of 3'.fwdarw.3' dinucleotide ester as a by-product (see Tetrahedron, 34, 1978, p. 3143).
There have been developed various methods for eliminating the foregoing problems. First, Reese et al. examined a phosphorylating agent carrying two kinds of leaving groups X and Y which differ in ability as a leaving group from one another (ability as a leaving group: X&gt;Y), as a result found out a phosphorylating agent of the formula [3] in which one of the leaving groups is substituted with p-nitrophenoxy group and investigated the application thereof to oligonucleotide synthesis. However, it can be applied only to the stepwise synthesis and thus it is still insufficient from the viewpoint of general-purpose properties of the method (Tetrahedron Letters, 30, 1978, p. 2727) ##STR3##
In addition, a method in which a trivalent phosphorylating agent is employed has been developed on the basis of studies of nucleic acid synthesis. This method comprises forming an internucleotide bond by the use of the high reactivity of the trivalent phosphorus atom and then oxidizing the trivalent phosphorus atom into a pentavalent phosphorus atom to form a triester compound. In the early stage of such studies, the use of phosphorochloridite in the reaction had been investigated and it was found out that an intended product can very rapidly be prepared (Tetrahedron Letters, 21, 1980, p. 719). However, this method likewise suffers from various problems that formation of the by-product, 3'.fwdarw.3' dinucleotide ester, is liable to occur, that the trivalent phosphorus atom is extremely unstable to moisture and that it is impossible to form any block bonding of the trivalent phosphorus atom. Therefore, this method is not practically applicable to a liquid phase method.
Then, Caruthers and his coworkers investigated the reaction in more detail and as a result, found out that morpholidite and diisopropylamidite are well applicable to the reaction (Nucleic Acid Research, 11, 1983, p. 2575; and J. Am. Chem. Soc., 105, 1983, p. 661). After the condensation reaction, the intended protected phosphoric acid triester can be obtained by oxidizing the condensate with iodine-water in a good yield. The nucleic acid synthesis through this amidite method has presently been variously improved from the viewpoint of carriers and reaction conditions and almost established as a routine work. Automatic nucleic acid synthesizers in which the reaction operations are mechanically performed have already been put on the market.
In addition, Boom et al. tried to apply this amidite method to synthesis of nucleopeptides with remarkable success (Tetrahedron, 44, 1988, p. 6515). Moreover, the phosphite method can be applied to phospholipid synthesis and there have been proposed synthesis of some phospholipids and derivatives thereof (see, for instance, Tetrahedron Letters, 29, 1988, p. 3631; and J. Org. Chem., 51, 1986, p. 2368).
However, the methods using a trivalent phosphorylating agent comprises complicated operations. For instance, the method using a chloridite requires the use of a very low reaction temperature of the order of -78 .degree. C. to suppress the formation of undesirable symmetrical by-products, while the amidite method requires the use of severe anhydrous conditions. For this reason, the phosphite method suffers from various problems if it is applied to the preparation of phospholipids in a practical scale, unlike the synthesis of nucleic acids in which it is necessary to prepare them in only a small amount.
On the other hand, there has recently been discovered a pentavalent phoshorylating agent where the phosphorus atom is activated by a substituent other than a chlorine atom and which behaves as a monofunctional agent though it is bifunctional, and it has practically been used.
First, Katagiri, Narang et al. developed phosphoroditriazolide of the formula [4] obtained by activating phosphorus atom with an azole instead of a chlorine atom and thus succeeded in the preparation of asymmetrical phosphoric acid esters without the formation of the undesirable by-product, 3'.fwdarw.3' dinucleotide esters (J. Am. Chem. Soc., 97, 1975, p. 7332). ##STR4##
In addition, in 1981, Boom et al. proposed the use of another useful phosphorylating agent, i.e., 1-hydroxybenzotriazole (HOBt) ester of the formula [5] (Tetrahedron Letters, 22, 1981, p. 3887). According to Boom et al., this method is likewise applicable to the formation of internucleotide bonds and synthesis of phospholipids and nucleopeptides, and it is capable of providing intended products in a high yield. However, this method requires a tremendous labor to remove the released 1-hydroxybenzotriazole and thus there remains a problem of operability. ##STR5##
Kunieda et al. proposed the use of a phosphorylating agent of the formula [6] which is substituted with 2-oxazolone (Tetrahedron Letters, 21, 1987, p. 2375). They find out that the phosphorylation with the agent of the formula [6] is promoted by the presence of a catalytic amount of metal ions (used in the form of an acetyl acetonate) and that the activation degree varies depending on the kinds of the metals used. They succeeded in the preparation of asymmetric phosphoric acid esters by making use of the foregoing fact, i.e., by differentiating the activation degree between the first and second phosphorylation reactions. However, this method is still insufficient from the viewpoint of simplicity and practicability. ##STR6##
Furthermore, Ramirez et al. develop a cyclic enediol derivative of the formula [7] (they call it "Cyclic EnedioI Phosphoryl (CEP) derivative") having a protective group which also serves as an activating group, and propose a method for forming asymmetric phosphodiester bonds from two different kinds of alcohols (Synthesis, 1985, p. 449). ##STR7##
As has been discussed above, most of these conventional methods for preparing compounds carrying asymmetric phosphodiester bonds suffer from a severe problem of formation of the undesirable symmetrical by-products. Moreover, these methods require the use of strict anhydrous and/or cryogenic conditions, a tremendous labor is required for preparing necessary reagents and the resulting reagents have low storage stability. Therefore, these methods are not always practicable.